Protocol 19328: End Point Analysis Assay - Ins2<Akita>

B6.Cg-Ins2^Akita Bdkrb2^tm1Jfh/SmiJ

Stock No: 006860

Version 5.0

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

HET = C/T
MUT = T/T
WT = C/C

 

>chr7:142678732-142678788 57bp GCAGAAGCGTGGCATTGTAG AGCTGGTAGAGGGAGCAGAT

Sequence

 

GCTCTTCTCTATCCAGCTAGAAGCATCAGGGGTGGGGGTCATGATCCTCCCGGACAACT
CAGGGTGGGGGTTATGGGCTACCTGACGGTATTGGTTATCTTGGGGAAAGGAACTGTCT
ATGTTCCAGGTTTGGTCTAACAGGTACCTGTGGGAGGTGCCCAGGGAGCCAGGAGAAA
ACTGGGGATGCAGAGGGAACCAAAGAAGTCTTGGGTAGATCTTTTAGGGGCTATTGTGG
CTAGGCAGATAGATAGATTAAGCTGTGGCTACCCTACCAGCCCCACTCATGTCTCCTGGT
CCCACATATGCACATGCACATACACACACAACTTGTGCTCATTCAAAGGTTTTATTCATT
GCAGAGGGGTAGGCTGGGTAGTGGTGGGTCTAGTTGCAGTAGTTCTCCAGCTGGTAGA
GGGAGCAGATGCTGGTG(C/T)AGCACTGATCTACAATGCCACGCTTCTGCTGGGCCACC
TCCAGTGCCAAGGTCTGAAGGTCACCTGCTCCCGGGCCTCCACCCAGCTCCAGTTGTG
CCACTGCCAGGGAGGTTGTGTCAGAGCAGGCCAGGGCATCAGCAGCACAGAAGCAAA
GACACATAGGAAAATCCAGTCAGGAGTAGCTGAGCAACCTCCTACTACCCCAGGTTTTC
TCGCCCCTTAACCCCTGCAGGTGGCAGGGGTTGCATGCCTGTGGGACAAGTCACTGGTC
ATGGATGGACACATGGGCCTCTAAAGCCTACTCATCTTCAGTCAGTGCAGTGCTCTATGA
GGGCCCTAAATAGGTACCTATCCTGGGATCTAGTCACATGTCCCAAGTTACTACCACCAA
GGACCCCTTAAGGCAGGATTCTTACTCATTCCTGGCAGCTGCCATGTTTACATATGTCTAG
CTTCCCCCAGAAACGTGTCCCCACTCCCTGGTTATCCCGAGACCCTCCACACCTAGGACA
CAATTTACAATGTCCCAAGTGTGAAGAAAACCAGGGTAGTTAGCACTGGGGACAG

 

Single Point Mutation: In the mutant allele a transition from G to A at nucleotide 1907 disrupted an Fnu4HI site in exon 3. This mutation changed the seventh amino acid in the A chain of mature insulin, Cys96 (TGC), to Tyr (TAC). The authors predict that the transition would disrupt a disulfide bond between the A and the B chains and would likely induce a major conformational change in insulin 2 molecules. RT-PCR studies suggest that both normal and mutant Ins2 alleles are transcribed similarly in pancreatic islets of heterozygous mice, although immunofluorescence and immunoblot analyses of heterozygous islets detected reduced levels of insulin and proinsulin.

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
13606	Fluorophore-1	GCA GAA GCG TGG CAT TGT AG	Quencher-1	Forward	A
13607	Fluorophore-2	AGC TGG TAG AGG GAG CAG AT	Quencher-2	Reverse	A
13608	Fluorophore-3	TCA GTG CTG CAC CAG C	Quencher-3	WT Probe
13609	Fluorophore-4	ATC AGT GCT ACA CCA GC	Quencher-4	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
13606	0.40 uM
13607	0.40 uM
Wt Probe	0.15 uM
Mutant Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--
4		--	repeat steps 2-3 for 40 cycles
5	40.0	--	Forever

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number	Strain Name
007688	129S6.B6-Ins2^Akita/CofJ
012307	B6.129-Apoe^tm3(APOE*4)Mae Ldlr^tm1(LDLR)Mae/J
006860	B6.Cg-Ins2^Akita Bdkrb2^tm1Jfh/SmiJ
006580	B6.Cg-Ins2^Akita Ldlr^tm1Her/J
004369	B6.Cg-Rag1^tm1Mom Ins2^Akita/J
003548	C57BL/6-Ins2^Akita/J
007562	D2.B6-Ins2^Akita/MatbJ
006867	FVB.B6-Ins2^Akita/MlnJ
008659	NOD.Cg-Rag1^tm1Mom Ins2^Akita Prf1^tm1Sdz/SzJ
014568	NOD.Cg-Rag1^tm1Mom Ins2^Akita Il2rg^tm1Wjl/SzJ
10 strains use this protocol